The identification of highly potent and broadly neutralizing antibodies isolated from HIV-1- infected donors suggests that when presented with a suitable antigenic structure, the human immune system can produce protective antibodies that HIV-1 is unable to evade. Understanding these antigenic structures is a primary objective in developing a vaccine capable of generating broadly protective humoral immunity. Most of these broadly neutralizing MAbs bind conformationally complex epitopes on Envelope that have been difficult to epitope map, and an increasing number of such MAbs are being identified using more efficient approaches to MAb isolation. However, the ability to characterize MAbs has not kept up, leaving a major gap between the growing ability to isolate relevant MAbs and the ability to molecularly define the immunogenic structures that gave rise to them. The goal of this proposal is to develop tools to rapidly and comprehensively map MAb epitopes on structurally complex viral Envelope proteins. Identifying the epitopes of potent and broadly neutralizing MAbs will enable Env variants exhibiting improved antigenic characteristics to be more rapidly designed and tested as vaccine candidates. PUBLIC HEALTH RELEVANCE: This project will contribute to human health by identifying the epitopes of potent and broadly neutralizing MAbs so that Env variants exhibiting improved antigenic characteristics can be more rapidly designed and tested as vaccine candidates.